US3573295A

US3573295A - Process for the preparation of 7-aminocephalosporanic acid

Info

Publication number: US3573295A
Application number: US741301A
Authority: US
Inventors: David Aaron Johnson; Elwin J Richardson; John Mckenna Roubie; Herbert Horatius Silvestri; Richard Root Smith
Original assignee: Bristol Myers Co
Current assignee: Bristol Myers Co
Priority date: 1968-07-01
Filing date: 1968-07-01
Publication date: 1971-03-30
Anticipated expiration: 1988-03-30
Also published as: CY825A; MY7500292A; KE2580A; GB1283223A

Abstract

7-AMINOCEPHALOSPORANIC ACID, A VALUABLE INTERMEDIATE FOR THE PREPARATION OF SEMI-SYNTHETIC CEPHALOSPORINS, IS PREPARED BY A PROCESS COMPRISING THE CONSECUTIVE STEPS OF: (A) ACYLATING CEPHALOSPORIN C BROTH WITH AN ISOCYANATE TO PRODUCE A COMPOUND CALLED N-(N''-ALKYL- OR ARYLCARBAMOYL)CEPHALOSPORIN C AND HAVING THE FORMULA

2-(HOOC-),3-(CH3-COO-CH2-),7-(HOOC-CH(-NH-CO-NH-R)-

(CH2)3-CO-NH-)-2-CEPHEM

IN WHICH R IS (LOWER)ALKYL OR ARLY; (B) RECOVERING DERIVATIVE III BY SOLVENT EXTRACTION; (C) SILYLATING THE CARBOXYL FUNCTIONS OF COMPOUND III TO FORM SILYL ESTERS; (D) HALOGENATING THE SILYL ESTER OF COMPOUND III TO PRODUCE AN IMINO-HALIDE; (E) FORMING AN INIMO-ETHER FROM THE IMINO-HALIDE BY TREATMENT WITH AN ALCOHOL; AND (F) MIXING SAID IMINO-ESTER WITH WATER OR AN ALCOHOL TO PRODUCE 7-AMINOCEPHALOSPORANIC ACID (7-ACA).

Description

United States Patent ABSTRACT OF THE DISCLOSURE 7-aminocephalosporanic acid, a valuable intermediate for the preparation of semi-synthetic cephalosporins, is prepared by a process comprising the consecutive steps of: (A) acylating cephalosporin C broth with an isocyanate to produce a compound called N-(N-alkylor arylcarbamoyl)cephalosporin C and having the formula I II I S now-p-wmM-c-rz l 0 NH ll 1 O N CH2OCCHs I :0 a 17111 0on1 R (III) in which R is (lower)alkyl or aryl;

(B) recovering derivative III by solvent extraction;

KC) silylating the carboxyl functions of compound III to form silyl esters;

(D) halogenating the silyl ester of compound III to produce an imino-halide;

(E) forming an imino-ether from the imino-halide by treatment with an alcohol; and

(F) mixing said imino-ether with water or an alcohol to produce 7-aminocephalosporanic acid (7-ACA).

BACKGROUND OF THE INVENTION (1) Field of the invention This invention relates to a novel process for the in situ preparation, subsequent harvesting and conversion of cephalosporin C derivatives into 7-ACA.

It is thus an object of the present invention to provide a new and improved process for the preparation of 7-ACA.

(2) Description of the prior art Recovery methods for cephalosporin C from fermentation broth are described in US. Pats. Nos. 3,093,638 and 3,094,527 wherein the processes described involve adsorption of the cephalosporin C onto an adsorbant as compared to solvent extraction. While (lower) alkylcarbamoylor arylcarbamoyl-cephalosporin C derivatives are not described as being formed in situ in cephalosporin C fermentation broth, or as being useful to aid in the extraction of cephalosporin C from its broth, or as being useful as a starting material in the production of 7-ACA, some of these compounds are described in the patent literature (e.g. British Pat. 1,064,495 and US. Pat. 3,227,712) as antibiotics. Several processes for the chemical cleavage of cephalosporin C or certain of its derivatives are described in the patent literature (U.S. Pats. Nos. 3,188,311, 3,234,223, 3,124,576 and British Pat. 1,041,985). None of these processes employ an N-(lower)alkylor arylcarbamoyl-cephalosporin C and a silyl ester thereof as a starting material.

ice

SUMMARY OF THE INVENTION This invention relates to and has for its object the provision of an improved process for the in situ preparation and recovery of a cephalosporin C derivative, said derivative being utilizable in a chemical cleavage, as its silyl ester, to 7-aminocephalosporanic acid.

7-aminocephalosporanic acid (7-ACA) is a most valuable intermediate in the preparation of a multitude of semi-synthetic cephalosporanic acid antibacterial agents. In view of the impracticability of the total synthesis of 7-ACA on a large scale, commercial supplies of the compound are prepared by the chemical degradation of naturally occurring cephalosporanic acids, i.e., cephalosporin C, which is produced by fermentation. Most 7-ACA is derived from cephalosporin C (U.S. Pat. 3,093,638) which has the structure The production of 7-ACA by currently available methods is fraught with difficulties from the fermentation to the chemical cleavage of cephalosporin C. Low yields of 7-ACA have made it difficult for cephalosphoranic acids to take their rightful place in antibiotic therapy. For this reason the processes of the present invention are a significant improvement over the methods of the prior art.

Cephalosporin C is characterized by an amino-acid function in its side chain. The amino-acid exists in the form of a zwitterion in aqueous solution and as such is very water soluble. Because of its highly ionic nature, it is extremely difiicult to harvest by solvent extraction of the fermentation broth. The harvesting procedure currently used involves the adsorption of the crude cephalosporin C from the fermentation broth onto a suitable adsorbant, i.e., charcoal, an ion exchanger resin, or the like, followed by elution, concentration and precipitation at the isoelectric point or by salt formation (U.S. Pat. No. 3,094,527). The low degree of efiiciency coupled with the complexity of each step in this process combine to make it very difiicult to manufacture 7-ACA.

The building block of the semi-synthetic cephalosporins is 7-ACA, whose structure is shown below:

HgN

l l O C 02H (II) Most 7-ACA is prepared by partial degradation of cephalosporin C or its derivatives by chemical means (U.S. Pats. Nos. 3,124,576, 3,188,311 and 3,234,223).

Again, most of these methods provide commercial yields that are undesirably low. Furthermore, these methods invariably employ as the starting material a purified form of cephalosporin C Which is diflicult to obtain.

It was therefore an object of the present invention to improve on the harvestable yields of cephalosporin C from its fermentation broth in such a chemical form as to be directly useable in subsequent cleavage reactions to produce 7-ACA Without substantial purification procedures.

The object of the present invention has been achieved by the provision, according to the present invention, of the process for the in situ preparation and harvesting of a derivative of cephalosporin C having the formula (III wherein R is (lower)alkyl but preferably ethyl, n-propyl, isopropyl, n-butyl or isobutyl; or a group of the formula wherein n is an integer of to 6 and R and R are alike or different and each is H, Cl, Br, F, N0 (lower)alkyl or (lower)alkoxy, but preferably hydrogen; which comprises the consecutive steps of:

(A) Adding an isocyanate having the formula in which R is as defined above, to a previously filtered and acid-incubated fermentation broth containing cephalosporin C, in a ratio of at least 2 moles of isocyanate per mole of cephalosporin C, but preferably in a ratio of about 2 to 10 moles of isocyanate per mole of cephalosporin C, and most preferably 5 to 8 moles of isocyanate per mole of cephalosporin C, at a pH of about 7 to 9, but preferably about 8, at a temperature in the range of about 20 to 60 C., but preferably in the range of about -5 C. to about 20 C. to form said derivative of cephalosporin C; and

(B) Recovering said derivative of cephalosporin C preferably by extraction using a water-immiscible organic solvent such as methyl isobutyl ketone, butanol, ethyl acetate or the like, but preferably butanol, at a pH of about 1 to 3, but preferably about pH 2.

The term (lower)alkyl for the purpose of the present invention is defined as an alkyl group comprised of 1 to 10 carbon atoms, including for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, etc., and the like, but especially methyl, ethyl, n-propyl, isopropyl, n-butyl and isobutyl. The terms (lower) alkoxy and halo(lower)alkyl are also defined as moieties containing 1 to 10 carbon atoms.

The fermentation of the mold known as cephalosporium, or a mutate thereof, produces a mixture of compounds, predominately cephalosporin C and lesser amounts of cephalosporin N (now known to be a penicillin). Cephalosporin C is acid stable while cephalosporin N is not. Accordingly, prior to the reaction of the fermentation broth with an isocyanate, it is desirable to destroy in situ any co-produced cephalosporin N. This is accomplished by acidification to about pH 2 with a mineral acid such as hydrochloric acid, sulfuric acid, phosphoric acid, or the like, followed by an incubation period of about 2 to 20 hours. The fermentation broth may be filtered before or after acidification and incubation.

Following this step, the filtrate-fermentation broth containing the cephalosporin C is adjusted to about pH 7 to 9, but preferably'about 8, by the addition of an alkali metal base such as sodium or potassium hydroxide.

The volume of the prepared broth is increased about 25% by the addition of acetone. The isocyanate is added to this solution with stirring in a ratio of at least 2, preferably about 2 to 10 moles of isocyanate per mole of cephalosporin C (broth concentration having been predetermined) but more preferably in a ratio of about 5 4 to 8 moles. The pH is held constant during the slow addition and for at least 30 minutes following the addition. The temperature of the broth is maintained below 40 C., but preferably at 0 C. to 15 C. during this time. The reaction is usually complete one hour after the addition is completed.

One-half volume of an immiscible organic solvent is added, preferably n-butanol, and the pH is adjusted to about 1 to 3, but preferably 2. The mixture is stirred and the organic solvent phase containing the cephalosporin C derivative III is collected.

The organic solution is concentrated in vacuo to about one-third its original volume at a temperature not to exceed 40 C. The concentrate is cooled to about 20 C. to 30 C. and the sodium salt of the cephalosporin derivative III is precipitated by the addition of a slight excess of sodium 2-ethylhexanoate dissolved in n-butanol. The mixture is cooled to 0 C. to 5 C. for 3 to 4 hours and the solid sodium salt of derivative III is collected by filtration. The filter cake is washed with cold butanol followed by an n-hexane washing. The product is dried in vacuo at about 60 C.

It is another object of the present invention to provide a superior process for the preparation of 7-ACA which is capable of using the compounds having Formula III without the necessity of preliminary purification or chemical conversion to cephalosporin C.

This object of the present invention has been achieved by the provision according to the present invention, of the process for the preparation of 7-aminocephalosporanic acid which comprises the consecutive steps of:

(A) Mixing a compound having the formula MO 0 (3 (CH) (i l /S l I 0 NH GIN n orn-o-o-om f I IIIH 002M R (III) in which each M is selected from the group consisting of hydrogen, metal and amine cations, and R is (lower) alkyl, but preferably ethyl, n-propyl, isopropyl, n-butyl and isobutyl; or a group of the formula wherein n is an integer of 0 to 6 and R and R are alike or different and each is H, Cl, Br, F, N0 (lower)alkyl or (lower)alkoxy, but preferably hydrogen; with at least two equivalents of a silyl compound of the formula f ins... R5 l R X or r /Si /R Ill 1 R NSi H Rs --N wherein R is hydrogen or (lower)alkyl and R is hydrogen, (lower)alkyl or but preferably dimethyldichlorosilane or trimethylchlorosilane; under anhydrous conditions in the presence of an excess of an acid deactivating tertiary amine selected from the group consisting of triethylamine, dimethylaniline, quinoline, lutidine, pyridine, or the like, in an inert solvent selected from the group consisting of methylene chloride, dichloroethane, chloroform, tetrachloroethane, nitromethane, diethylether, or the like, to produce the corresponding silyl di-ester of compound III;

(B) Mixing said silyl ester with a halogenating agent selected from the group consisting of phosphorous pentachloride, phosphorous pentabromide, phosphorous trichloride, phosphorous tribromide, oxalyl chloride, p-toluenesulfonyl halide, phosphorous oxychloride, phosgene, or the like, in a molar ratio of 2 or more moles of halogenating agent per mole of silyl ester, but preferably about two moles, under anhydrous conditions in an inert solvent such as methylene chloride, dichloroethane, chloroform, tetrachloroethane, nitromethane, diethyl ether or the like, in the presence of an acid deactivating tertiary amine selected from the group consisting of triethylamine, dimethylaniline, quinoline, lutidine, pyridine or the like, at temperatures below C., but preferably in the range of 40 C. to 60 C., to produce in solution the corresponding imino-halide;

(C) Mixing with said solution of imino-halide an alcohol selected from the group consisting of aliphatic alcohols having 1 to 12 carbon atoms and phenylalkyl alcohols having 1 to 7 alkyl carbon atoms, at a temperature below 0 C., but preferably -40 C. to 70 C. to produce in the solution the corresponding imino-ether; and

(D) Mixing said solution of imino-ether under acidic conditions with water or an aliphatic alcohol, or a mixture of both, to produce 7-aminocephalosporanic acid.

The process of the invention unexpectedly produces high yields under both laboratory and commercial scale conditions. The yields, which may be in the order of 50% to 70%, are attributed to the use of silyl esters on the carboxyl groups of the compounds of Formula III. The silyl esters may be prepared and hydrolyzed to the acid again Without the loss of product, especially if the reaction proceeds at the temperatures below C., preferably -40 C. to -70 C., during formation of the imino-ether. Moreover, the use of silyl esters rather than the esters employed in the previously cited patents simplifies the process since the silyl ester hydrolyzes simultaneously with the splitting of the double bond of the imino-group and avoids the separate step of splitting the 4 carboxylic esters of the prior art processes.

The formation of the silyl ester is accomplished by reacting a silyl compound of Formula IV or V, under anhydrous conditions, in an inert organic solvent, with the compound of Formula III, or a salt thereof, in the presence of an acid deactivating tertiary amine.

Suitable inert solvents include amongst others methylene chloride, dichloromethane, chloroform, tetrachloroethane, nitromethane, benzene and diethyl ether.

Suitable salts of compound III include amongst others alkali metal and alkaline earth metal salts such as potassium, sodium, calcium, aluminum etc. Also acceptable are ammonium and amine salts, preferably tertiary amines such as triethylamine, dibenzylamine, trimethylamine, N-methylmorpholine, pyridine, N benzyl B phenethylamine, 1 ephenamine, N,N' dibenzylethylene diamine, dehydroabietylamine, N-(lower)-alkylpiperidines such as N-ethylpiperidine, and the like. Tertiary-amine salts are preferable.

Suitable acid deactivating tertiary amines include amongst others triethylamine, dimethylaniline, quinoline, lutidine, pyridine, etc. The quantity of acid deactivating amine used is preferably an amount equivalent to about 75% of the total acid generated in the process by the halogenating agent and the halosilane compound reacting with compound III.

Suitable silyl compounds of Formulas IV and V are: trimethyl chlorosilane, hexamethyl disilazane, triethyl chlorosilane, methyl trichlorosilane, dimethyl dichlorosilane, triethyl bromosilane, tri-n-propyl chlorosilane, bromomethyl dimethyl chlorosilane, tri-n-butyl chlorosilane, methyl diethyl chlorosilane, dimethyl ethyl chlorosilane, phenyl dimethyl bromosilane, benzyl methyl ethyl chlorosilane, phenyl ethyl methyl chlorosilane, triphenyl chlorosilane, triphenyl fluorosilane, tri-o-tolyl chlorosilane, tri-p-dimethylaminophenyl chlorosilane, N-ethyl triethyl silylamine, hexaethyl disilazane, triphenyl silylamine, tri-n-propyl silylamine, tetraethyl dimethyl disilazane, etc. The same effect is produced by hexa-alkyl phenyl disilazane, hexaphenyl disilazane, hexa-p-tolyl disilazane, etc. The same effect is produced by hexaalkylcyclotrisilazanes or oeta-alkylcyclotetrasilazanes. Other suitable silylating agents are silylamides and silylureides such as trialkylsilylacetamide and a bis-trialkylsilylacetamide.

The imino compound is preferably an imino chloride or bromide which can be prepared by reacting the silyl ester of compound III with a halogenating agent such as phosphorus pentachloride, phosphorus pentabromide, phosphorus trichloride, phosphorus tribromide, oxalyl chloride, p-toluene sulfonic acid halide, phosphorus oxychloride, phosgene, etc., under anhydrous conditions in the presence of acid binding agents at temperatures preferably below 0 C. such as 40 to 60 C.

A very important step for high yields of the process of the present invention is the formation of the imino ether by reacting the imino halide under anhydrous conditions with a primary or secondary alcohol at temperatures between 20 and 70 C., preferably about 40 C. to 70 C. Temperatures higher than -40 C. result in a substantial reduction in yield.

Suitable alcohols for producing the imino ethers from the imino-halides are primary and secondary alcohols having the general formula R OH in which R is selected from the group consisting of (A) (lower)alkyl, having 1 to 12 carbon atoms, preferably having 1-3 carbon atoms, such as methanol, ethanol, propanol, isopropanol, n-butanol, amyl alcohol, decanol, etc.; (B) phenylalkyl having 1 to 7 alkyl atoms, such as benzyl alcohol, 2- phenylethanol-l, etc.; (C) cycloal-kyl ,such as cyclohexylalcohol, etc; (D) hydroxyalkyl having 2 to 12 carbon atoms, preferably at least 3 carbon atoms, such as 1,6- hexanediol, etc.; (E) alkoxyalkyl having 3 to 12 carbon atoms, such as 2-methoxyethanol, 2-isopropoxyethanol, 2-butoxyethanol, etc.; (F) aryloxyalkyl, having 3 to 7 carbon atoms in the aliphatic chain, such as 2-p-chlorophenoxyethanol, etc.; (G) aralkoxyalkyl, having 3 to 7 carbon atoms in the aliphatic chain, such as 2-(p-methoxybenzyloxy)-ethanol, etc.; (H) hydroxyalkoxyalkyl, having 4 to 7 carbon atoms, such diglycol. Also mixtures of these alcohols are suitable for forming the imino ethers.

After formation of the imino-ether from the iminohalide, the imino bond must be split to produce 7-ACA. The process is accomplished by mild hydrolysis or alcoholysis. If the quantity of acid deactivating tertiary amine present in the process is a quantity less than the acid produced by the silylation and halogenation, the cleavage will probably proceed simultaneously upon completion of the formation of the imino-ether. If however the quantity of acid deactivating amine was more than the acid produced, the cleavage step will require the careful addition of a quantity of H+ to effect the cleavage.

The 7-ACA is harvested from the reaction mixture by adjusting the pH of the mixtures to or near the isoelectric point of the 7-ACA, following which the 7-ACA crystallizes and is collected by filtration.

For optimum yields of 7-ACA, it is preferred to use high concentrations of the reactants. For example, in the formation of the silyl esters, a 20% to 30%, preferably 25% by Weight of compound III is suspended in an inert organic solvent and a base for the best results. The silane is employed preferably in excess, i.e. 10% to 60%, above theoretical amounts.

One molecule of cephalosporin C derivative HI possesses two reactive carboxyl groups capable of forming silyl esters. Therefore, in terms of the silylation reaction, one mole of III is equal to two equivalent weights.

Accordingly, when compound III is treated with dichlorodimethylsilane, one molecule of compound III (two equivalent weights) is treated with at least one molecule (two equivalent weights) of dichlorodimethylsilane. Similarly, when compound III is treated with chlorotrimethylsilane, one molecule of compound I II (two equivalent weights) is treated with at least .two molecules (two equivalent weights) of chlorotrimethylsilane.

This enables the use of solvents which are not absolutely dry because trace amounts of water are removed therefrom by reacting with the excess silylating agent. Obviously the quantity of the silane compound required is dependent upon whether one or both carboxyl groups of the compound III are available for silyl ester formation. The reaction scheme is illustrated below:

8 DESCRIPTION OF THE PREFERRED EMBODIMENTS Example l.--In situ preparation of N-(N'-butylcarbamoyl)cephalosporin C Twenty-nine hundred liters of whole fermentation broth containing cephalosporin C was mixed with 108 kg. of a filtering aid and 300 ml. of silicone antifoam' and then filtered at pH 6.9 at 10 C.

Suflicient oxalic acid was added to the filtered broth to make the pH 3.1. Following this addition, the pH was adjusted to pH 2 by the addition of 30% sulfuric acid. Fresh filtering aid, 54 kg., was added and the mixture was filtered. The tfiltrate thus obtained was extracted with /2 volume of methyl isobutyl ketone (MIBK) at pH 2 and then separated. The MIBK phase was discarded.

One-fourth volume of acetone was added to the extracted broth and the pH was adjusted to 7.85 with a solution of sodium hydroxide. n-Butylisocyanate (2"kg./ 1000 l. of broth) was added slowly with stirring (a molar The following examples will serve to illustrate but not to limit the scope of the present invention.

ratio of about 6.1 moles of n-butylisocyanate per mole of cephalosporin C).

The pH was constantly maintained at pH 7.8 to 8.0 by the addition of 15% sodium hydroxide and the temperature was held in the range of C. to C. during the addition. Stirring was continued until the pH remained constant without the addition of further sodium hydroxide, a time of about 2 hours.

The pH was adjusted to pH 2 by the addition of 30% sulfuric acid and the acylated broth was extracted with three-fourths volume of n-butanol. The butanol phase was water-washed and then concentrated to approximately one-half the starting volume at 36 C. in vacuo. The water content of the concentrate was negligible.

The concentrate was cooled and a solution of sodium ethylhexanoate in n-butanol was added to pH 4.8. The sodium salt of N-(N-butylcarbamoyl)cephalosporin C precipitated and was collected by filtration. The precipitate was washed with butanol, re-slurried in petroleum ether, washed with acetone, filtered and dried in a vacuum oven at 40 C. to 60 C. to yield the product. Assay of the spent broth indicated the presence of less than cephalosporin C activity. The product was of adequate purity for the subsequent preparation of 7-ACA. It was analyzed as the diacid.

Analysis.--Calcd for C H N O S-H O (percent): C, 47.36; H, 6.06; N, 10.54; H O, 3.38. Found (percent): C, 47.85; H, 6.52; N, 10.54; S, 6.45; H O, 3.48.

Example 2.-Preparation of 7-aminocephalosporanic acid Sodium N-(N-butylcarbamoyl)cephalosporin C [13.4 grams], 45 milliliters of methylene chloride, 1.0 ml. of dimethylaniline and 3.67 ml. of triethylamine were mixed together. Dichlorodimethylsilane (5.0 ml.) was added with stirring at a temperature of 28 C. The solution was stirred minutes. The solution was then cooled to 60 C. and 11.5 grams of phosphorus pentachloride dissolved in 100 ml. of methylene chloride was added while the temperature of the reaction was kept below C. An additional 12.4 ml. of N,N-dimethylaniline in 10 ml. methylene chloride was added. The temperature was held below 40 C. for 2 hours, then chilled to 73 C., and a mixture of 60 ml. of methanol and 2.5 ml. dimethylaniline chilled to --78 C. was added slowly. The temperature rose to 47 C. The mixture was stirred and recooled to '50 C. At the end of two hours, 55 ml. of water heated to +95 C. was added. The temperature rose to +5 C. The mixture was cooled in an ice bath and stirred for 4 minutes. The pH was 01. Ammonium hydroxide (21.5 ml.) was added over 8 minutes to a pH of 3.8. The mixture was stirred several hours and then filtered. The precipitate was collected, washed with 25 ml. of methylene chloride, then 25 ml. of water, then 50 ml. of methanol and finally 50 ml. of' acetone. The solid was dried to yield 4.05 grams of 93.7% 7-ACA.

Example 3.-In situ preparation of sodium N-(N- phenylcarbamoyl)cephalosporin C Substitution in the procedure of Example 1 for the 6.111 molar ratio of n-butylisocyanate:cephalosporin C used therein of a 7:1 molar ratio of phenylisocyanate produced a satisfactory product for cleavage to 7-ACA. Assay of the spent broth after extraction indicated the presence of less than 15% cephalosporin C activity. It was assayed as the diacid.

Analysis.-Calcd for C H N O S (percent): C, 51.68; H, 4.90; N, 10.48; S, 6.00. Found (percent): C, 51.63; H, 5.27; N, 10.62; S, 6.14.

Example 4.Preparation of 7-aminocephalosporanic acid Sodium N-(N-phenylcarbamoyl)cephalosporin C [13.9 g.], milliliters of methylene chloride, 1 ml. of dimethylaniline and 3.67 ml. of triethylamine were mixed together. Dichlorodimethylsilane (5.5 ml.) was added with stirring at a temperature of 25 28 C. The slurry was stirred for 75 minutes, then cooled to -60 C. and 12.0 grams of PCl in 100 ml. of methylene chloride was added. An additional 11.0 ml. of dimethylaniline in 10 ml. of methylene chloride was added. The temperature was maintained at 40 C. for 2 hours. The solution was cooled to -75 C. and a solution of 60 ml. methanol and 2.5 ml. dimethylaniline cooled to 78 C. was added. The temperature rose to 52 C. The temperature was held in the range of 45 C. to -50 C. for 2 hours. Fifty ml. of water warmed to C. was added with stirring. The temperature rose to +5 C. After 4 minutes of stirring (pH 0.0), 22 ml. of NH OH was added over an 8 minute interval to pH 3.7. The slurry was stirred for several days with cooling. The precipitated 7-ACA was collected by filtration, washed with 50 ml. methylene chloride, 40 ml. water, 50 ml. methanol, then 50 ml. acetone.

Example 5.In situ preparation of sodium N-(N- benzylcarbamoyl)cephalosporin C Substitution in the procedure of Example 1 for the 61:1 molar ratio of n-butylisocyanate:cephalosporin C used therein of a 7.011 molar ratio of benzylisocyanate per mole of cephalosporin C produces sodium N-(N benzylcarbamoyl) cephalosporin C.

Example 6.7-ACA from sodium N-(N'-benzylcarbamoyl)cephalosporin C Substitution in the procedure of Example 2 for the sodium N (N butylcarbamoyl)cephalosporin C used therein of sodium N-(N'-benzylcarbamoyl)cephalosporin C produces 7-ACA.

Example 7.Sodium N-(N-((lower))alkyl-or arylcarbamoyl)cephalosporin C derivatives Substitution in the procedure of Example 1 for the n-butylisocyanate used therein of and Example 8 Substitution in the procedure of Example 2 for the sodium N (N' butylcarbamoyl)cephalosporin C used therein of the carbamoylcephalosporin C derivatives prepared in Example 7 produces 7-ACA.

While in the foregoing specification various embodiments of this invention have been set forth in specific detail and elaborated for the purpose of illustration, it will be apparent to those skilled in the art that this invention is susceptible to other embodiments and that many of the details can be varied widely without departing from the basic concept and the spirit and scope of the invention.

1 1 We claim: 1. The process for the in situ preparation and harvesting of a carbamoyl derivative of cephalosporin C having the formula wherein R is (lower)alkyl or a group of the formula wherein n is an integer of to 6 and R and R are alike or different and each is H, Cl, Br, F, N0 (lower)alkyl or (lower) alkoxy; which comprises the consecutive steps of:

(A) adding an isocyanate having the formula in which R is as defined above, to an acid-incubated fermentation broth containing cephalosporin C, which was previously prepared by fermentation of a mold of the cephalosporium genus, in a ratio of at least 2 moles of isocyanate per mole of cephalosporin C, at a pH above 7, at a temperature below 40 C., to form said carbamoyl derivative of cephalosporin C; and (B) recovering said carbamoyl derivative of cephalosporin C from the fermentation broth by extraction with a water-immiscible solvent. 2. The process of claim 1 for the in situ preparation and harvesting of a carbamoyl derivative of cephalosporin C having the formula wherein R is (lower)alkyl or a group of the formula wherein n is an integer of 0 to 6 and R and R are alike or different and each is H, Cl, Br, F, N0 (lower)alkyl or (lower)alkoxy; which comprises the consecutive steps of:

'(A) adding an isocyanate having the formula in which R is as defined above to a previously filtered and acid-incubated fermentation broth containing cephalosporin C, which was previously prepared by fermentation of a mold of the cephalosporium genus, in a ratio of about 2 to moles of isocyanate per mole of cephalosporin C, at a pH of about 7 to 9, at a temperature in the range of about 0 C. to about 25 C. to form said carbamoyl derivative of cephalosporin C; and

(B) recovering said carbamoyl derivative of cephalosporin C by extraction using a water-immiscible organic solvent at a pH in the range of about 1 to 3.

r 12 3. The process of claim 1 for the in situ" preparation and harvesting of a carbamoyl derivative of cephalosporin C having the formula wherein R is (lower)alkyl or a group of the formula wherein n is an integer of 0 to 6 and R is H, Cl, Br, F, N0 (lower) alkyl or (lower) alkoxy; which comprises the consecutive steps of:

(A) adding an isocyanate having the formula in which R is as defined above, to a previously filtered and acid-incubated fermentation broth containing cephalosporin C, which was previously prepared by fermentation of a mold of the cephalosporium genus, in a ratio of about 5 to 8 moles of isocyanate per mole of cephalosporin C, at a pH of about 7 to 9, at a temperature of about 0 C. to 15 C., to form said carbamoyl derivative of cephalosporin C; and (B) recovering said carbamoyl derivative of cephalosporin C by extraction using a water-immiscible organic solvent selected from the group consisting of methyl isobutyl ketone, ethylacetate, butanol, chloroform, methylene chloride and dichloroethane, at a pH in the range of about 1 to 3. 4. The process of claim 1 for the in situ preparation and harvesting of a derivative of cephalosporin C having the formula wherein R is ethyl, n-propyl, isopropyl, n-butyl or isobutyl; or a group of the formula wherein n is an integr of 0 to 6; which comprises the consecutive steps of:

(A) adding an isocyanate having the formula in which R is as defined above, to a previously fil tered and acid-incubated fermentation broth containing cephalosporin C,, which was previously prepared by fermentation of a mold of the cephalosporium genus, in a ratio of about 6 moles of isocyanate per mole of cephalosporin C, at a pH of about 8, at a temperature of about 0 C. to about 5 C., to form said carbamoyl derivative of cephalosprin C; and

(B) recovering said carbamoyl derivative of cephalosporin C by extraction using n-butanol, at a pH of about 2.

3,573,295 13 14 5. The process for the preparation of 7-aminocephalotetrachloroethane, nitromethane or diethyl ether; in sporanic acid which comprises the consecutive steps of: the presence of an acid deactivating tertiary amine (A) mixing a compound having the formula such as trimethylamine, triethylamine, dimethylaniline, quinoline, lutidine or pyridine; at temperatures in the range of ---10 C. to 60 C.; to produce in H o H I II I S solution the corresponding imino-halide; MO2C (CH2) C N I 0 (C) mixing With said solution of imino-halide an al- IIH N OH 0 a CH cohol selected from the group consisting of aliphatic 0:0 V alcohols having 1 to 12 carbon atoms and phenyll 1 M 10 alkyl alcohols having 1 to 7 alkyl carbon atoms; at a temperature in the range of 20 C. to 70 C.; R to produce in the solution the corresponding iminoether; and

(D) mixing said solution of imino-ether under acidic conditions with water or an aliphatic alcohol or a mixture of both; at a temperature about 0 C.; to produce 7-aminocephalosporanic acid.

in which each M is selected from the group consisting of hydrogen, metal and amine cations, and R is (lower)alkyl or a group of the formula R2 6. The process of claim 5 for the preparation of 7- aminocephalosporanic acid which comprises the consecutive steps of:

(A) mixing a compound having the formula wherein n is an integer of O to 6 and R and R are alike or different and each is H, Cl, Br. F, N0 f ii f /S (lower)alkyl or (lower)alkoxy; 2 2)3 "ON:l I O with a silyl compound of the formula III}? 0 N CH2-O H 5 Y R4 /R4" 17H 002M SiNH R5 R (III) R NH l or RS1X in which each M is selected from the group consist- 1 ing of hydrogen, metal and amine cations, and R is ethyl, n-propyl, isopropyl, n-butyl or isobutyl; or a u R m 39 group of the formula wherein R R and R are selected from the group consisting of hydrogen, halogen, (lower)alkyl, halo(lower) alkyl and phenyl, vat least one of the said R R and R' groups being other than halogen or hydrogen; R is (lower)alkyl, m is an integer of 1 to 2 and X is selected from the group consisting of halogen and wherein n is an integer of 0 to 6 and R is H, Cl, Br, F, N0 (lower)alkyl or (lower)alkoxy; with a silyl compound of the formula R5 R R -N Si NH R5 4 R4 or Rfl S!iX wherein R is hydrogen or (lower)alkyl and R is a hydrogen, (lower)alkyl or R4 In R5 R5li wherein R R and R are selected from the group 1 consisting of hydrogen, halogen, (lower)alkyl, halo- (lower)alkyl and phenyl, at least one of the said R under anhydrous conditions; in a ratio of at least one R and R groups being other than halogen or hyequivalent or silylating agent per equivalent of comdrogen; R is (lower)alkyl, m is an integer of 1 to 2 pound III; in the presence of an acid deactivating terand X is selected from the group consisting of halotiary amine selected from the group consisting of trigen and ethylamine, trimethylamine, dimethylaniline, quinoline, lutidine and pyridine; in an inert solvent selected from the group consisting of methylene chloride, dichloroethane, chloroform, tetrachloroethane, R nitro-methane and diethyl ether; to produce the corresponding silyl ester of compound III;

(B) mixing said silyl ester with a halogenating agent selected from the group consisting of phosphorus pen- Wherein R is hydrogen or (lower)alkyl and R is hydrogen, (lower)alkyl or tachloride, phosporus pentabromide, phosphorus trichloride, phosphorus tribromide, oxalyl chloride, p- R Si toluenesulfonyl halide, phosphorus oxychloride and phosgene; in a molar ratio of 2 to 4 mole of halogenating agent per mole of silyl ester; under anunder anhydrous conditions in a ratio of about 1.2 hydrous conditions; in an inert organic solvent such to about 2 equivalents of silyating agent per equivas methylene chloride, dichloroethane, chloroform, alent of compound III, in the presence of an acid deactivating tertiary amine selected from the group consisting of trimethylamine, triethylamine, dimethylaniline, quinoline, lutidine and pyridine in an inert organic solvent selected from the group consisting of methylene chloride, dichloromethane, chloroform, tetrachloroethane, nitromethane and diethyl ether, to produce the corresponding silyl ester of compound III;

(B) mixing said silyl ester with a halogenating agent selected from the group consisting of phosphorus pentachloride and phosphorus oxychloride, in a molar ratio of 2 to 3 moles of halogenating agent per mole of silyl ester, under anhydrous conditions in methylene chloride, dichloroethane, chloroform or tetrachloroethane, in the presence of a tertiary amine such as triethylamine, dimethylaniline or pyridine, at temperatures in the range of 40 C. to -60 C., to produce in solution the corresponding irrnino-halide;

(C) mixing with said solution of imino-halide methanol, ethanol, n-propanol or isopropanol, at a temperature in the range of 40 C. to 70 C., to produce in the solution the corresponding iminoether; and

(D) mixing said solution of imino-ether under acidic conditions with water or an alcohol selected from the group consisting of methanol, ethanol, n-propanol or isopropanol, or a mixture thereof, at a temperature in the range of about 10 C. to about +10 C., to produce 7-aminocephalosporanic acid.

7. The process of claim 6 wherein R is ethyl, n-propyl, isopropyl, n-butyl, isobutyl, phenyl or phenethyl; the silyl compound is dimethyldichlorosilane or trimethylchlorosilane; the inert organic solvent is methylene chloride or dichloroethane; and the halogenating agent is phosphorus pentachloride and it is used in a molar ratio of two moles of phosphorus pentachloride per mole of silyl ester.

8.. The process for the in situ preparation and harvesting of a derivative of cephalosporin C and its subsequent cleavage to 7-aminocephalosporanic acid which comprises the consecutive steps of:

(A) adding to a previously filtered and acid incubated fermentation broth containing cephalosporin C 'which was previously prepared by fermentation of a mold of the cephalosporium genus an isocyanate having the formula wherein R is (lower)alkyl or a group of the formula wherein n is an integer of to 6 and R and R are alike or difierent and each is H, Cl, Br, F, N0 (lower)alkyl or (lower)alkoxy; in a ratio of about 2 to 10 moles of isocyanate per mole of cephalosporin C; at a pH of about 7 to '9; at a temperature in the range of about 0 C. to about 25 C.; to proin which R is as defined above;

(B) recovering said derivative of cephalosporin C by extraction using a water-immiscible organic solvent at a pH in the range of about 1 to 3;

(C) mixing the cephalosporin C derivative, or a salt thereof, with a silyl compound of the formula wherein R R and R are selected from the group consisting of hydrogen, halogen, (lower) alkyl, halo- (lower)alkyl and phenyl, at least one of the said R R and R groups being other than halogen or hydrogen; R is (lower)alkyl, m is an integer of 1 to 2 and X is selected from the group consisting of halogen and wherein R is hydrogen or (lower) alkyl and R is hydrogen, (lower)alkyl or R5 R 's i under anhydrous conditions, in a ratio of at least one equivalent of silylating agent per equivalent of compound III, in the presence of an acid deactivating tertiary amine selected from the group consisting of triethylamine, trimethylamine, dimethylaniline, quinoline, lutidine and pyridine, in an inert solvent selected from the group consisting of methylene chloride, dichloroethane, chloroform, tetrachloroethane, nitromethane and diethyl ether, to produce the corresponding silyl ester of compound HI;

(D) mixing said silyl ester with a halogenating agent selected from the group consisting of phosphorus pentachloride, phosphorus pentabromide, phosphorus trichloride, phosphor-us tribromide, oxalyl chloride, p-toluenesulfonyl halide, phosphorus oxychloride and phosgene, in a molar ratio of 2 to 4 moles of halogenating per mole of silyl ester, under anhydrous conditions in methylene chloride, dichloroethane, chloroform, tetrachloroethane, nitromethane, in the presence of trimethylamine, triethylamine, dimethylaniline, quinoline, lutidine or pyridine, at tempera tures in the range of -10 C. to -60 C. to produce in solution the corresponding imino-halide;

(E) mixing with said solution of imino-halide an alcohol selected from the group consisting of aliphatic alcohols having 1 to 12 carbon atoms and phenyl alkyl alcohols having 1 to 7 alkyl carbon atoms, at a temperature in the range of 20 C. to C. to produce in the solution the corresponding iminoether; and

(F) mixing said solution of imino-ether under acidic conditions with water or an aliphatic alcohol, or a mixture of both, at a temperature about 0 C., to produce 7-arninocephalosporanic acid.

9. The process of claim 8 which comprises the consecutive steps of:

(A) adding to a previously filtered and acid-incubated fermentation broth containing cephalosporin C which was previously prepared by fermentation of a mold of the cephalosporium genus an isocyanate having the fonrnula wherein R is n-propyl, n-butyl, isobutyl, or phenyl; 1n a ratio of about 6 moles of isocyanate per mole of cephalosporin C; at a pH of about 8; at a tempera- 1 7 1 8 ture of about C. to about C.; to produce a (D) mixing said silyl ester with phosphorus pentachloderivative of cephalosporin C having the formula ride, in a molar ratio of,2 to 3 moles of halogenating agent per mole of silyl ester, under anhydrous conditions in methylene chloride, chloroform, or dichloro- S ethane, in the presence of triethylamine, dimethyl- 5 aniline or pyridine, at temperatures in the range of llIH H C. to C., to produce in solution the cor- CH2 OT responding imino-halide; I l (E) mixing with said solution of imino-halide, meth- I COZH l0 anol, ethanol, n-propanol or isopropanol, at a tem- R 1 perature in the range of 40 C. to C. to

- produce in the solution the corresponding iminoin which R is as defined above; ether} f (B) recovering said derivative of cephalosporin C by (F) salfi solutlon 0f lmlno'ether under acldic extraction using mbutanol at a PH of about 15 conditions with Water or methanol, ethanol, n-pro- (C) mixing the cephalosporin C derivative, or a salt Pan01 OI {sopropanol or a mlxturfi thereof, at a thereof, with dimethyldichlorosilane or trimethylchlo- Pmamre m the range of about about rosilane under anhydrous conditions, in a ratio of to Produce 7'amlnocephalosporanlc acidabout 1.2 to 2 equivalents of silylating compound per equivalent of compound III, in the presence of 20 References Cited an acid deactivating group selected from the group UNITED STATES PATENTS consisting of trimethylamine, triethylamine, dimeth- 3,499 909 3/1970 Weissenburger et a1. 7 ylaniline, quinoline, lutidine and pyridine, in methyl- 3993:6323 6/1963 Abraham et aL 260 243'C ene chloride or dichloroethane, to produce the corresponding silyl ester of compound III; 25 NICHOLAS S. RIZZO, Primary Examiner